1. Field of the Invention
The invention relates to a system(s), method(s) and apparatus(es) for assessment of local microvasculature and microcirculatory vasoreactivity.
2. Description of the Related Art
Multiple studies have indicated that assessment of the microvasculature in an accessible area not only delineates the status of the local microvasculature at that site but also correlates with vascular injury throughout the body, including the heart. [Anderson T J: Assessment of endothelial function: research tool or clinical reality? Cardiology Review: Endothelial Function Forum CME, July 1999; Anderson T J, Meredith I T, Yeung A C, et al. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med 1995; 332:488; Anderson T J, Overhiser R W, Haber H, Charbonneau F. A comparative study of four anti-hypertensive agents on endothelial function in patients with coronary disease. J Am Coll Cardiol 1998; 31:327A, Abstract #1147 54.]However, the ability to interrogate local microvasculature in a safe, noninvasive, nonintrusive manner has been limited.
Prior to the development of the current invention, assessments of the effects of drugs and alternative agents (herein referred to interchangeably as “drugs”, “agents” or “study agents” at the level of the microvasculature (e.g., arterioles, capillaries, venules) were performed primarily in in vitro tissue baths, after systemic drug administration (intravenous, intramuscular, oral), or after driving drug through the skin with electric current.
In vitro testing of vasoactive drugs typically entails exposure of tissue to different concentrations of an agent (or alternative stimulus) by direct application or by immersion in a drug-containing bath. Such testing led to the discovery in the early 1980's of the importance of the microvasculature and the endothelium lining its vessels; Acetylcholine, a prominent neurotransmitter, causes vasodilation of blood vessels with an intact inner endothelial lining and vasoconstriction of vessels with damaged or missing endothelial lining. [Furchgott R F, Zawadzki J V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288:373-6.] While valuable for initial characterization of drug effect, this method is not readily applicable in intact humans and does not necessarily reflect what would happen in an intact preparation; i.e., the findings may not readily transfer to clinical settings. These limitations have prompted in vivo studies in healthy volunteers and patients.
The evaluation in intact humans primarily has been accomplished by systemic administration of a drug (e.g., by a pill, lozenge, solution, or systemic intravenous or intramuscular administration). Systemic administration typically generates direct and indirect responses throughout the body; i.e., at the level of the heart, arteries, arterioles, capillaries, venules and veins. Delineation of effects at the level of the microvasculature is limited by a variety of factors, including:                difficulty obtaining/titrating tissue levels;        difficulty comparing active and placebo effects concurrently;        activation of multiple systemic responses and reflexes which may modulate the effect at the target site;        the potential for unwanted/dangerous systemic effects and side effects, thereby making testing impractical or impossible. For example, the inventor and his colleagues have shown that testing the effects of nicotine on the microvasculature entailed the use of systemic doses (e.g., via lozenges) that resulted in pronounced changes in systemic blood pressure and heart rate that necessitated stopping the study in some subjects. [Mo C, Stout R G, Shelley K H, Tantawy H, Silverman D G: Acute microcirculatory effects of nicotine in non-smoking volunteers. Anesthesiology 2004; 101:A246.]. Likewise, to test the systemic effects of a vasoconstricting drug such as phenylephrine [Silverman D G, Stout R G: Distinction between atropine-sensitive control of microvascular and cardiac oscillatory activity. Microvasc Res 63:196-208, 2002], one may need to deliver doses of drug which can potentially cause significant systemic vasoconstriction and hypertension.        
To overcome the aforementioned limitations associated with systemic administration of a drug, investigators have employed selective intravascular injection into a regional artery or vein. Despite the undesirable features of this approach as summarized below, the perceived importance of determining the reactivity of the in different disorders has prompted clinicians and investigators to administer pharmacologic agents such as acetylcholine into coronary (heart) as well as brachial (arm) and femoral (leg) arteries. In the context of atherosclerotic disease, intracoronary injection of acetylcholine is associated with impaired vasodilation or frank vasoconstriction, leading to the suggestion that it serve as a test for early detection of coronary artery disease. Prexler H, Zeiher A M. Progression of coronary endothelial dysfunction in man and its potential clinical significance. Basic Research In Cardiology. 1991; 2:223-32., Bossaller C, Habib G B, Yamamoto H, Williams C, Wells S, Henry P D. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5′-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. Journal of Clinical Investigation. 1987; 79:170-4; and Thanyasiri P, Celermajer D S, Adams M R. Endothelial dysfunction occurs in peripheral circulation patients with acute and stable coronary artery disease. American Journal of Physiology Heart & Circulatory Physiology. 2005; 289.] Likewise, the vasodilatory response to brachial or femoral artery injection of acetylcholine is compromised in the presence of coronary artery disease. [Yoshida M, Imaizumi T, Ando S, Hirooka Y, Harada S, Takeshita A. Impaired forearm vasodilatation by acetylcholine in patients with hypertension. Heart & Vessels. 1991; 6:218-23] and hypertension. However, the need for an intra-arterial injection has led to such invasive tests being confined primarily to research settings.
Potential limitations of such regional injection include:                Invasiveness of injection.        Discomfort associated with injection and hence pain-related responses.        Regional pressure and volume changes as a consequence of injections.        The need to apply tourniquets to the regions and/or physically alter blood flow in an alternative manner.        Leakage to the other regions.Hence, such testing primarily has been confined to research settings.        
In an effort to avoid the invasive nature of such injections, investigators have sought other means of delivery. However, the techniques recommended heretofore have significant shortcomings. Most notably, local injection of drug at the planned site of monitoring can be painful and may produce unreliable results because of local tissue damage and irritation, as well as potential disturbance of tissue planes, thereby leading to inconsistent spread of drug. [Opazo Saez A M. Mosel F. Nurnberger J. Rushentsova U. Gossl M. Mitchell A. Schafers R F. Philipp T. Wenzel R R. Laser Doppler imager (LDI) scanner and intradermal injection for in vivo pharmacology in human skin microcirculation: responses to acetylcholine, endothelin-1 and their repeatability. British Journal of Clinical Pharmacology 59(5):511-519, 2005 May.]
This has led to the use of a needleless technique such as iontophoresis, wherein an electrical current drives drug through the skin. As discussed below in greater detail, there are iontophoretic devices that indeed have the ability for drug delivery as well as for laser Doppler monitoring of local blood flow. Such monitoring in the context of iontophoresis has quantified vasodilation at selected sites in response to iontophoretic application of acetylcholine in healthy subjects, and has documented that this response is compromised in patients with diabetes. However, iontophoresis is irritating to the tissue, such that the process itself alters microvascular function and microcirculatory flow; and iontophoretic delivery of inactive vehicle causes changes.
While one could wait several minutes after iontophoresis to allow for the effects of electrical current to abate, this also would allow time for the drug to redistribute and potentially leave the local area, especially if the drug has a vasodilatory effect which would increase its uptake by the circulation. Overall, factors limiting the widespread use of iontophoresis in the clinical setting include:                1) it can be painful (this not only would be undesirable for the subject but also might cause pain-induced changes in systemic blood flow);        2) the device requires a well or pad for the drug and is relatively bulky and thus is not necessarily well-suited for placement on small sites such as digits and also on sites that are not perfectly level; Moreover, these components may impede monitoring at portions of the delivery/study sites;        3) dosage and time-course are dictated by the need to deliver drug with an electric current;        4) if one has to check periodically to see the effect of iontophoretic delivery on local perfusion while allowing for the direct effect of the electrical stimulation to abate, this could lead to a time-consuming evaluation and undesirable probe repositioning;        5) the vehicle alone (without active drug) can cause changes in local perfusion;        6) iontophoresis can cause an electrical burn under the pad; and        7) electric current itself has been shown to be a potent vasodilator; even when data are modified to account for a potential current-induced effect, the current remains a potential confounder in such studies; its effect varies among specific vehicles and drugs as well as spatially over the area of drug delivery.        
The limitation as well as the potential benefits of iontophoresis in the context of disease have been studied extensively. [Christen S, Delachaux A, Dischl B, Golay S, Liaudet L, Feihl F, Waeber B. Dose-dependent vasodilatory effects of acetylcholine and local warming on skin microcirculation. Journal of Cardiovascular Pharmacology. 2004; 44:659-64. Noon J P, Walker B R, Hand M F, Webb D J: Studies with iontophoretic administration of drugs to human dermal vessels in vivo: cholinergic vasodilatation is mediated by dilator prostanoids rather than nitric oxide. Br J Clin Pharmacol 1998; 45:545-50; Wang S, Omar W, Awad A, Scannell M, Silverman D G: Direct and reflexive autonomic effects of acupuncture in healthy subjects. Int Anesth Res Soc S-215, 2002; Morris S J, Shore A C, Tooke J E. Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM. Diabetologia. 1337; 38:1337-44. Ledger P. Skin biological issues in electrically enhanced transdermal delivery. Advanced Drug Delivery Reviews. 1991; 9:289-307), Peters E J, Armstrong D G, Wunderlich R P, Bosma J, Stacpoole-Shea S, Lavery L A. The benefit of electrical stimulation to enhance perfusion in persons with diabetes mellitus. Journal of Foot & Ankle Surgery. 1998; 37:396-400.; Ferrell W R, Ramsay J E, Brooks N, Lockhart J C, Dickson S, McNeece G M, Greer I A, Sattar N. Elimination of electrically induced iontophoretic artefacts: implications for non-invasive assessment of peripheral microvascular function. Journal of Vascular Research. 2002; 39:447-55.; Droog E J, Sjoberg F. Nonspecific vasodilatation during transdermal iontophoresis—the effect of voltage over the skin. Microvascular Research. 2003; 65:172-8.; Khan F, Newton D J, Smyth E C, Belch J J. Influence of vehicle resistance on transdermal iontophoretic delivery of acetylcholine and sodium nitroprusside in humans. Journal of Applied Physiology. 2004; 97:883-7.; Mar and Holowatz L A, Thompson C S, Minson C T, Kenney W L. Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin. Journal of Physiology. 2005; 563:965-73].
Transdermal Drug Delivery:
The prior art includes transdermal applicants (e.g., gels, creams, ointments that are secured to a supporting substrate or backing)—herein referred to as “patches” because they commonly are supplied in the form of a patch—for a variety of purposes that transdermally deliver drugs in a non-iontopheretic manner (i.e., transdermal (or trans-surface) delivery without the use of electrical energy). However, these patches have limitations due to restricted size and restricted dose. Various patches have been used in the delivery of systemically effective plasma concentrations of a variety of drugs, including:                fentanyl patch for delivery of systemic levels of the analgesic opioid fentanyl, in order to achieve systemic levels commensurate with those by intravenous injection;        scopolamine patch to achieve systemic levels of scopolamine for the treatment of nausea;        nicotine patch to provide systemic levels of nicotine in individuals hoping to wean from cigarettes without symptoms of nicotine withdrawal;        nitroglycerin patch to achieve systemic levels of the cardiac medicine nitroglycerin;        clonidine patch to achieve systemic levels of this antihypertensive medication; and        estrogen patch to deliver systemic levels of this hormone.As noted above, each of the aforementioned patches is designed to deliver a systemic level of drug and thus, in its current form, is not suitable for the goals of strictly local assessment of the microvasculature. If one of these “systemic patches” were to be used for the purpose of assessing the effect of the drug, it would be plagued with most of the limitations and systemic side effects associated with oral or intravenous administration—e.g., remote effects, systemic effects.        
Prior patch preparations have been designed to achieve a local effect (e.g., to treat local pain, itching, etc.). These include ointments, many of which are in the form of patches:                Lidocaine ointment/patch—delivers lidocaine to a painful site under the patch in the hope of achieving local pain relief.        Tiger Balm® ointment/patch (for local muscle pain).        TheraPatch® (with calamine to treat local itching).        EMLA cream: eutectic mixture of the local anesthetics lidocaine and prilocaine (which is used to provide analgesia in children prior to needle insertion).        
Prior preparations used to achieve a local vascular effect include:                Phenylephrine nose spray to achieve vasoconstriction of nasal mucosa.        Nitroglycerin ointment and l-arginine ointment to increase local blood flow and thereby improve local healing (based on known vasodilating properties of the drug).        
To the best of the inventor's knowledge, none of these patches has been introduced or utilized for the diagnosis and assessment of vascular disorders. In addition, except for the local effect of EMLA (“eutectic mixture of local anesthetics”), they are designed to cause changes to a relatively large area; e.g., 5×5 cm size as opposed to customized limited doses. This can result in significant systemic levels of the drug and thus their use for the assessment of changes in the microvasculature would be compromised. Furthermore, the widespread local delivery will affect much more than the local microvasculature and may encompass multiple vascular beds, including larger arteries and veins. An additional problem of a large delivery area is that it may promote “steal” among vessels in the region as each of the dilated vessels “competes” for increased blood flow.
Monitoring the Local Microvasculature:
As stated above, none of the preparations to date has been designed to include medications for the purpose of measuring microvascular responsiveness; nor have they been adapted for real-time monitoring while the patch is in place. They thus have not been adapted with any mechanism for assessing such flow and have not been accompanied by a placebo patch for the purpose of enabling effective double-blind investigations which isolate the effect of the drug itself as opposed to other variables such as placebo patch application and administration of the vehicle for drug delivery. Additionally, the preparations often are not designed for consistently optimum light transmission, with customization of agent concentration and vehicle color and consistency.
Although multiple mechanisms for assessing surface blood flow have been developed, none of these current mechanisms for assessing surface blood flow is ideally suited for monitoring the changes in perfusion induced by micro-patches in accordance with the present invention and as described below in greater detail. In particular, the shortcomings of the current mechanisms for assessing surface blood flow are as follows:                Thermometry may be more indicative of flow in large vessels than in a restricted microcirculatory bed; the small temperature change that may result from a local microvascular effect induced by a micro-patch is overshadowed by other changes. Furthermore, it is influenced by ambient temperature.        Angiography requires radiological contrast material and special detection equipment; moreover, it is relatively ineffective at delineating relatively small changes in the microvasculature.        Radioisotopes entail the use of radioactive material.        Capillaroscopy requires a relatively cumbersome microscope and is not suited to monitoring multiple sites.        Plethysmography primarily is reflective of volume changes in a region, as opposed to local microcirculatory changes.        Laser Doppler flowmetry, with a surface probe or via a scanning imager, and laser speckle analysis appear better suited for monitoring the microvasculature as developed in accordance with the present invention and as discussed below in the description of the present invention.        As stated above in the context of iontophoresis, the local microcirculation may be monitored with a light-transmitting technique such as laser Doppler flowmetry. This technique entails delivery of light of uniform wavelength to the tissue under study; this is scattered (phase-shifted) by moving red blood cells near the tissue surface, such that the degree of alteration is proportional to the concentration of moving blood cells and their velocity. As with any of the monitoring techniques that rely upon light transmission, laser Doppler flowmetry would be distorted by surface application of a nontransparent agent or vehicle as required by iontophoresis and/or their opaque carrying structures.        In its simplest form, laser Doppler flowmetry entails placements of a probe on a study site to gain an indication of blood. However, since a laser Doppler reading is influenced by the number and size of blood vessels under a probe (commonly between 20 and 50 capillaries in one to three arteriolar-capillary networks), the reading is relative. Therefore, the technique is primarily used for comparison among sites or to monitor changes at a given site during a systemic challenge (since the probe can remain at a single monitoring site without the need for removal and replacement). Without the inventive modifications as discussed below in describing the present invention, use of a standard laser Doppler probe with a standard drug patch is prone to error and distortion, most notably: a) most patches have an opaque background, thereby preventing light transmission to the study site; b) after baseline readings are obtained, the probe would need to be removed to enable patch placement—this leads to distortion as a consequence of spatial heterogeneity (as discussed below). The inventor and his colleagues have documented variability among sites within millimeters of each other. [Braverman I M. Schechner J S. Silverman D G. Keh-Yen A. Topographic mapping of the cutaneous microcirculation using two outputs of laser-Doppler flowmetry: flux and the concentration of moving blood cells. Microvascular Research 44(1):33-48, 1992 July].        The laser Doppler scanning imager scans multiple sites to provide an image of a broader region. However, since it monitors sites sequentially, it does so at the cost of decreased temporal resolution at a given site and hence has the potential for increased temporal artifact (as a consequence of changing conditions as well as subject movement).        The laser speckle imager overcomes the laser Doppler scanner's lack of temporal resolution by acquiring signals simultaneously on a multipixel charged couple device. However, it is not certain as to whether it can generate a successful signal/noise ratio and may not enable optimum sensitivity to gradations in flow; and the speckle pattern is very sensitive to surface irregularities as may result from drug and/or vehicle application; moreover, the degree of speckling may change with time as a cream or ointment is absorbed (even without changes in blood flow).        
In view of the foregoing, a need exists for improved diagnostic testing systems, methods and apparatuses for integrated use in the assessment of microcirculation. The present invention provides such systems, methods and apparatuses.